Supporting InformationFauconnier et al. 10.1073/pnas.0908540107SI Materials and MethodsECG Recording and Analysis. Mice were monitored by ECG per-formed with a signal transmitter-receiver (RPC-1; DSI) connectedto a data acquisition system (IOX2; EMKA Technologies). Thedata were collected continuously over 24 h at a sampling rate of1,000Hz. Continuous digital recordings were analyzed offline usingthe ECG-auto, version 1.5.12.22 (EMKA Technologies). Tele-metric data were scanned using software by blinded observers tomeasure heart rate variability (HRV) parameters, as describedpreviously (1). In brief, ECG signals were digitally filtered between0.1 and 1,000 Hz and analyzed manually to detect arrhythmiasidentified by RR intervals two times greater or smaller than themean, and then quantified. RR intervals were calculated fromnocturnal ECG recordings.HRV was analyzed similarly on the same recordings.For HRV

analyses, the RR values not included between RR ± 2 SD (95.5%confidence intervals) were removed from the analyses. Theywere not replaced by any averaged or interpolated beat (2). Theindexes used were the mean RR interval and SDNN, which re-flects total autonomic variability.

RyR2 and NOS Biochemistry.Hearts fromWT andmdx age-matchedlittermates were lysed isotonically in 1.0 mL of a buffer containing50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 20 mM NaF, 1.0 mMNa3VO4, and protease inhibitors. The levels of nNOS, eNOS, andiNOS and were compared between mdx and WT muscle homo-genates by immunoblot analysis. Heart homogenates (50 μg) wereseparated by 10% PAGE, and the immunoblots were preparedusing antibodies against nNOS, eNOS, and iNOS (all 1:2,000 di-lution; BD Biosciences). All immunoblots were developed andquantified using the Odyssey infrared imaging system (LICORBiosystems) and infrared-labeled secondary antibodies.An anti-RyR antibody (4 mg; 5029 Ab) or the NOS isoform

antibodies (4mg;BDBiosciences)wereused to immunoprecipitateRyR2, nNOS, eNOS, or iNOS from 500 mg of heart homogenate.Thesampleswereincubatedwiththeappropriateantibodyin0.5mLof a modified RIPA buffer [50 mMTris-HCl (pH 7.4), 0.9%NaCl,5.0 mM NaF, 1.0 mM Na3VO4, 1% Triton- X100, and proteaseinhibitors] for 1 h at 4°C. The immune complexes were incubatedwith protein G Sepharose beads (Sigma-Aldrich) at 4°C for 1 h,afterwhich the beadswerewashed three timeswith buffer. Proteinswere separated on SDS/PAGE gels (6% for RyR2; 10% for NOSenzymes) and transferred onto nitrocellulose membranes for 1 h at200 mA (SemiDry transfer blot; Bio-Rad). Immunoblots wereprepared as described above. RyR2 S-nitrosylation was measuredby developing immunoblots with both a RyR antibody (AffinityBioreagents) and an anti–Cys-NO antibody (Sigma-Aldrich).

Enzymatic Cell Dissociation. Cardiac ventricular myocytes wereenzymatically dissociated using standard procedures (3). In brief,the mouse was euthanized by cervical dislocation, after which theheart rapidly was excised and retrogradely perfused at 37°C for6–8 min with a modified tyrode solution [113m NaCl, 4.7 mMKCL, 0.6 mM KH2PO4, 0.6 mM Na2HPO4, 1.2 mM MgSO4,12 mM NaHCO3, 10 mM KHCO3, 10 mM Hepes, 30 mMTaurine (pH 7.4)] containing 0.1 mg·mL−1 of liberase (Roche).Isolated myocytes were then transferred to the same enzyme-freesolution containing 1 mM CaCl2.

Measurement of Intracellular Ca2+ and Ca2+ Sparks.Cells were loadedfor 30 min at RT with fluo-4 AM (5 μmol/L; Molecular Probes),then field-stimulated at 1 Hz with 1-ms current pulses delivered

via two platinum electrodes, one on each side of the perfusionchamber. Changes in fluo-4 AM fluorescence were recorded usingan LSM510 Meta confocal microscope equipped with a 63× wa-ter-immersion objective (numerical aperture: 1.2; Zeiss). Allmeasurements were performed in line-scan mode (1.5 ms/line),and scanning was carried out along the long axis of the cell. Anexcitation wavelength of 488 nm was used, and emitted light wascollected through a 505-nm long-pass filter. The laser intensityused (3%–6% of the maximum) had no noticeable deleteriouseffect on the fluorescence signal or cell function over the courseof the experiment. To enable comparisons between cells, thechange in fluorescence (ΔF) was divided by the fluorescence de-tected immediately before the 0.5-Hz stimulation pulse (F0).Spontaneous Ca2+ sparks were recorded in quiescent cells fol-lowing 5-min stimulations to reach steady-state SR-Ca2+ content.

Calcium Imaging. To quantitatively monitor intracellular Ca2+

concentration, cardiomyocytes were loaded for 30 min at RT with10 μM indo-1 AM (Invitrogen), then field-stimulated at 1 Hz (20V, 1 ms), and simultaneously illuminated at 305 nm using a xenonarc bulb light. Indo-1 AM fluorescence emitted at 405 nm and 480nm was recorded simultaneously using IonOptix acquisitionsoftware (Hilton). To record spontaneous Ca2+ waves in theresting condition, stimulation was stopped for 30 s once a Ca2+

transient steady-state was reached. Indo-1 AM calibration wasperformed as described previously (4), using the following equa-tion:

�Ca2þ

� ¼ Kd × β× ðR−RminÞ=ðRmax −RÞ;where R is the fluorescence ratio (F405/F480), Rmin is the

fluorescence ratio in the absence of [Ca2+], Rmax is the fluo-rescence ratio in the presence of saturating [Ca2+], Kd is thedissociation constant, and β is the ratio of the F480 values at 0and saturating level of Ca2+.

Measurement of Membrane Potential. Action potentials (APs) wererecorded in rod-shaped Ca2+-tolerant myocytes under current-clamp conditions using a whole-cell patch-clamp technique at 22 ±2 °C with an Axopatch 200B (Axon Instruments), as describedpreviously (5). Pipettes (2–3 MΩ) were filled with a recordingsolution [130 mM KCl, 25 mM Hepes, 3 mM ATP(Mg), 0.4 mMGTP(Na), 05 mM EGTA, pH adjusted to 7.2 with KOH]. Themyocytes were superfused with Tyrode’s solution (135 mM NaCl,1 mM MgCl2, 4 mM KCl, 11 mM glucose, 2 mM Hepes, 1.8 mMCaCl2, pH adjusted to 7.4 with KOH) (6). APs were elicited by0.2-ms current injections of suprathreshold intensity. Signals wereacquired at 10 KHz and filtered at 5 kHz using low-pass Besselfilter. Cells were stimulated routinely at 0.1 Hz until APs stabilized(3–5 min). Once steady state was established, myocytes werepaced at 5 Hz for 1 s every 5 s, to record DADs. Data acquisitionand analyses were performed using Pclamp version 10.1 (AxonInstruments). Resting membrane potential (RMP) and AP dura-tion (APD) at 20% (APD20), 30% (APD30), 50% (APD50), 90%(APD90), and 95% (APD95) of repolarization were measured.

Statistical Analysis. Statistical comparison were performed withSigmastat version 3.5 (Systat Software). For spatiotemporalproperties of Ca2+ sparks, normality tests failed, and thus datawere statistically compared using Kruskal-Wallis one-way AN-OVA on ranks. Other statistical comparisons were done withANOVA. Data are expressed as mean ± SEM, and differenceswere considered significant at P < 0.05.

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1. Thireau J, et al. (2008) Increased heart rate variability in mice overexpressing the Cu/Znsuperoxide dismutase. Free Radic Biol Med 45:396–403.

2. Thireau J, Zhang BL, Poisson D, Babuty D (2008) Heart rate variability in mice: Atheoretical and practical guide. Exp Physiol 93:83–94.

3. Fauconnier J, et al. (2007) Effects of palmitate on Ca(2+) handling in adult control andob/ob cardiomyocytes: impact of mitochondrial reactive oxygen species. Diabetes 56:1136–1142.

4. Grynkiewicz G, Poenie M, Tsien RY (1985) A new generation of Ca2+ indicators withgreatly improved fluorescence properties. J Biol Chem 6:3440–3450.

5. Fauconnier J, et al. (2005) Frequency-dependent and proarrhythmogenic effects of FK-506 in rat ventricular cells. Am J Physiol Heart Circ Physiol 288:H778–H786.

6. Fauconnier J, Bedut S, Le Guennec JY, Babuty D, Richard S (2003) Ca2+ current-mediated regulation of action potential by pacing rate in rat ventricular myocytes.Cardiovasc Res 57:670–680.

Fig. S1. Treatment with NAC prevents RyR2 S-nitrosylation and depletion of calstabin2 in mdx hearts. RyR2 was immunoprecipitated from heart homogenateof 35-day-old mdx mice and WT littermates. In addition, a group of 3-week-old mdx mice were treated for 2 weeks with NAC. Immunoblots were prepared forRyR2, S-nitrosylation of cysteine residues on RyR2 (Cys-NO), and calstabin2 bound to RyR2. The blots are representative of three independent experiments.

Fig. S2. Effect of NAC on RyR function in mdx mice. Ventricular cardiomyocytes were isolated from 35 day-old mdx mice (n = 3) and separated into twobatches. One batch was maintained in a normal external buffer, and the other was maintained for 1 h before use in experiments in the same mediumcontaining the ROS/RNS scavenger NAC (20 mM). Cells were then loaded for 30 min with fluo-4 AM, with fluorescence monitored by laser scanning confocalmicroscopy. RyR2-induced diastolic SR Ca2+ leak was estimated based on the average frequency of sparks. The high sparks frequency observed in mdx cells wasreversed to a value comparable to that in WT cells (Fig. 2D). *P < .05 mdx vs. NAC-treated mdx. n = 651 sparks in 30 cells in mdx; n = 175 sparks in 30 cells inNAC-treated mdx.

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Fig. S4. Isoproterenol-induced ectopic Ca2+ transients in mdx mice. Isolated cardiomyocytes were paced at 1 Hz. Ca2+ fluorescence was recorded in line-scanmode (x vs. time; 1.5 ms/line) using confocal microscopy. (A) Typical sequences of Ca2+ transients obtained in cardiomyocytes after acute application of 100 nMisoproterenol. (B) This treatment significantly increased the number of ectopic Ca2+ transients, which were rare in WT and S107-treated mdx mice. Data areexpressed as mean ± SEM. *P <.05 WT vs. mdx ;#P <.05 mdx vs. S107-treated mdx.

Fig. S3. Consequences of SR Ca2+ leak on AP duration and RMP. APs were recorded on isolated cardiomyocytes from 35-day-old mice using the patch-clamptechnique with an internal medium containing a low EGTA concentration to avoid excessive Ca2+ buffering, which could interfere with Ca-dependent reg-ulatory mechanisms on RMP. APs were recorded at 5 Hz. The stimulation was interrupted to challenge resting membrane potential and occurrence of DADs.(A–C) Typical train of APs followed by a rest period recorded in mdx cardioyocytes (A), S107-treated mdx cardiomyocytes (B), and cardiomyocytes incubatedwith 20 mM NAC (C, Left). The arrow in A indicates a DAD in themdx cardiomyocytes. This behavior was not observed after S107 treatment or NAC incubation.The first APs recorded in a train are shown in the right panel in an expanded time scale. AP durations were measured at different phases of AP repolarization.(D) Data summary showing a shortening effect of both S107 treatment and NAC incubation that appear to be significant at 90% and 95% repolarization, asdenoted by the arrow in A (Right). RMP depolarization occurring in mdx cardiomyocytes was prevented by S107 treatment or NAC incubation. Data areexpressed as mean ± SEM. n = 15 in mdx (black), n = 11 in S107-mdx (red), and n = 10 in NAC-mdx (blue). *P < 0.05 mdx vs. S107-treated mdx; #P < 0.05 mdx vs.NAC-treated mdx.

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